Chef mapper xa pulsed field electrophoresis system

Yeast strains were propagated in YPM at 20 °C to an OD600 >1 and then harvested by centrifugation (3,000×g, 5 min, 4 °C). Supernatants were decanted, and cells were resuspended in 10 ml of 4 °C 50 mM EDTA (pH 8). Cell concentrations were determined with a Nucleocounter^® YC-100™ (ChemoMetec) and 1.2 × 10⁸ cells were placed in each sample plug. Sample plugs were prepared with the CHEF Genomic DNA Plug Kit for Yeast (Bio-Rad) according to the manufacturer’s instructions.
Sample plugs were loaded into the wells of a 1.0 % pulse field certified agarose (Bio-Rad) gel. PFGE was performed at 14 °C in 0.5× TBE buffer [89 mMTris, 89 mM boric acid, 2 mM EDTA (pH 8)]. A CHEF Mapper XA pulsed field electrophoresis system (Bio-Rad) was used with the following settings: 6 V/cm in a 120° angle, pulse length increasing linearly from 26 to 228 s, and total running time of 38 h. A commercial chromosome marker preparation from S. cerevisiae strain YNN295 (Bio-Rad) was used for molecular mass calibration. After electrophoresis, the gels were stained with ethidium bromide and scanned with Gel Doc XR+ imaging system (Bio-Rad).

High-molecular-weight genomic DNA (>50 Kb) extraction and purification were performed using the Genomic-tip 100/G genomic DNA isolation kit (Qiagen). DNA concentration was measured by using NanoDrop (Thermo Fisher Scientific) and Qubit 2.0 (Invitrogen) instruments. The integrity and quality of genomic DNA were confirmed by using the Bio-Rad^® CHEF Mapper^® XA Pulsed Field Electrophoresis system. High-quality sequencing data were generated using four different sequencing and mapping technologies (i.e., Illumina, 10X Chromium, PacBio Sequel, and DLS BioNanoSaphyr optical mapping). Illumina sequencing libraries were constructed by using the NEBNext UltraTM DNA Library Prep Kit (Illumina). The 10X Chromium genomic libraries were prepared by using the Chromium Genome HT Library Kit and Gel Bead Kit v2. The SMRTbell library was prepared using SMRTbell Express Template Preparation Kit. For the construction of optical genome maps, standard BioNano protocols, nicking, labeling, repair, and staining processes were implemented. Specifically, DNA was digested by the single-stranded nicking endonuclease Nt.BspQI. Optical maps were assembled with BioNano Irys View analysis software; only single molecules with a minimum length of 100 kb and six labels per molecule were used.

The strains genetic relatedness was observed by DNA macrorestriction with 30 U of ApaI enzyme (New England BioLabs Inc., United States) followed by pulsed-field gel electrophoresis (PFGE), as previously described (Durmaz et al., 2009 (link)). The electrophoresis was run with 0.5X TBE buffer at 14°C, with 6 V/cm², for 20 h and linear ramping, with initial and final switch times of 5 and 30 s, respectively, using a CHEF Mapper system (CHEF Mapper XA Pulsed Field Electrophoresis System—Bio-Rad Laboratories Inc., United States). After electrophoresis, the gel was stained with 0.01% SYBRSafe (Invitrogen, Life Technologies, United States) in 0.5x TBE buffer for 1 h, followed by visualization using the ChemiDoc XRS (Bio-Rad Laboratories Inc., United States).
We compared the DNA band profiles using the Bionumerics software (version 7.6, Applied-Maths, Belgium) with 0.5% optimization and 1.25% tolerance parameters, with cluster analysis done by the unweighted pair group method using the arithmetic average (UPGMA). Isolates with 100% similarity by PFGE were considered indistinguishable and clustered in the same pulsotype and subtype; isolates with similarity 80% were considered closely related and clustered in the same pulsotype, but different subtypes; isolates with similarity <80% were considered possibly related and are classified as belonging to different pulsotypes.

To separate plasmids from chromosomal DNA, S1 nuclease pulsed-field gel electrophoresis (PFGE) was performed according to Barton et al. with modifications [12 (link)]. The total DNA in agarose gel plugs was incubated in distilled water for 30 min at 20°C. Then, the plugs were incubated with 30 U of S1 nuclease (TaKaRa, Shiga, Japan) for 30 min at 4°C, followed by incubation for 40 min at 37°C. Reactions were stopped by incubation for 5 min at 20°C with 0.5 M EDTA. The plugs were rinsed using 10 mM Tris-HCl/1 mM EDTA (pH 8.0) and incubated for 1 h at 4°C with 0.5 × TBE buffer (50 mM Tris, 45 mM boric acid, and 0.5 mM EDTA). The total DNA digested with S1 nuclease in plugs was loaded onto a 1% agarose gel and run with a CHEF Mapper XA pulsed-field electrophoresis system (Bio-Rad, Hercules, CA, USA) at 14°C in 0.5 × TBE buffer for 20 h under the following conditions: auto algorithm, 20-kb low molecular weight (MW), 250-kb high MW, and 6.0 V/cm. After electrophoresis, the agarose gel was stained with SYBR Gold nucleic acid gel stain (1/10,000 dilution; Thermo Fisher Scientific) for 1 h at 20°C. Bands were visualized using a WSE-5200A Printgraph 2M system (ATTO, Tokyo, Japan). The visualized bands were collected and stored at −30°C until whole-genome analysis was performed.

Yeast chromosome integrity was analyzed as described previously [168 (link)] with certain modifications. Yeast cells grown at 23°C were embedded in 20-μl plugs of low-melting-point SeaKem Gold agarose (Lonza, Basel, Switzerland). The plugs were digested with Zymolyase 100T (BioShop) overnight at 37°C with gentle rotation and then with proteinase K (A&A Biotechnology) and RNase A (Sigma-Aldrich, St. Louis, MO, USA) overnight at 37°C with gentle rotation. Then, the plugs were treated with 2 U or 5 U of S1 nuclease (Thermo Scientific) for 30 min in S1 buffer provided by the manufacturer. Next, plugs were placed in the wells of a 1% D5 agarose gel (BioMaxima, Lublin, Poland) in 1x TAE and sealed with the same agarose. Electrophoresis was performed on a CHEF Mapper XA pulsed-field electrophoresis system (Bio-Rad, Hercules, CA, USA) for 18 h in 1x TAE buffer at 6 V/cm and 12°C, angle of 120°, and switch time of 60–85 s, with a ramp-up of 0.8. After electrophoresis, the DNA was stained with 0.5 μg/ml ethidium bromide (Sigma-Aldrich) and visualized using UV light.